Hepatocyte growth factor (HGF/SF) is a polypeptide identified and purified by Nakamura, T., et al., Biochem. Biophys. Res. Commun. 22 (1984) 1450-1459. It was further found that hepatocyte growth factor is identical to scatter factor (SF), Weidner, K. M., et al., Proc. Natl. Acad. Sci. USA 88 (1991) 7001-7005. HGF is a glycoprotein involved in the development of a number of cellular phenotypes including proliferation, mitogenesis, formation of branching tubules and, in the case of tumor cells, invasion and metastasis. For a status review, see Stuart, K. A., et al., Int. J. Exp. Pathol. 81 (2000) 17-30.
Both rat HGF and human HGF have been sequenced and cloned (Miyazawa, K. et al., Biochem. Biophys. Res. Comm. 163 (1989) 967-973; Nakamura, T., et al., Nature 342 (1989) 440-443; Seki, T., et al., Biochem. and Biophys. Res. Comm. 172 (1990) 321-327; Tashiro, K., et al., Proc. Natl. Acad. Sci. USA 87 (1990) 3200-3204; Okajima, A., et al., Eur. J. Biochem. 193 (1990) 375-381).
HGF is a protein with high similarity to human plasminogen (38% amino acid sequence identity). HGF and plasminogen are both synthesized as a single chain polypeptide which is proteolytically processed to a disulfide-linked heterodimer. HGF contains an N-terminal domain four consecutive kringle domains and a carboxyterminal, protease-like domain. Different truncated HGF variants have been described. NK1 is the shortest HGF variant described. NK1 contains amino acids 32-210 and is truncated after the first kringle domain (Lokker, N. A., and Godowski, P. J., J. Biol. Chem. 268 (1993) 17145-17150). NK2 consists of the N-terminal amino acid terminus and kringle 1 and kringle 2 and is the naturally occurring product of an alternatively spliced HGF mRNA (Chan, A. M., et al., Science 254 (1991) 1382-1385). Further HGF variants containing parts of the heavy chain of HGF (amino acids 1-494, containing the alpha-subunit of HGF from amino acids 1-463) are described by Lokker, N. A., EMBO J. 11 (1992) 2503-2510).
It was further found that an HGF/SF fragment, termed NK4, consisting of the N-terminal hairpin domain and the four kringle domains of HGF/SF has pharmacological properties that are completely different from those of HGF/SF, and is an antagonist to the influence of HGF/SF on the motility and the invasion of colon cancer cells, and is, in addition, an angiogenesis inhibitor that suppresses tumor growth and metastasis (Parr, C., et al., Int. J. Cancer 85 (2000) 563-570; Kuba, K., et al., Cancer Res. 60 (2000) 6737-6743; Date, K., et al., FEBS Lett. 420 (1997) 1-6; Date, K., et al., Oncogene 17 (1989) 3045-3054).
NK4 is prepared according to the state of the art (Date, K., et al., FEBS Lett. 420 (1997) 1-6) by recombinant expression of HGF cDNA in CHO cells and subsequent digestion with pancreatic elastase. Two other isoforms of HGF (NK1 and NK2) encoding the N-terminal domain and kringle 1, and the N-terminal domain and kringles 1 and 2, respectively, were produced in E. coli (Stahl, S. J., Biochem. J. 326 (1997) 763-772). However, this method results only in about an amount of HGF-derived proteins which is about 10-20% of the total protein.